The long-term goal of this project is to improve the quality of epidermal stem cells (EpiSCs) for therapeutic transplantation, stem cell (SC)-directed gene therapy and for the treatment of degenerative skin disorders by elucidating both intrinsic and extrinsic mechanisms regulating EpiSC proliferative potential. We previously demonstrated that the transcription factor p63 is essential for the proliferative potential of EpiSCs. However, dual promoters in the p63 gene drive expression of TA isoforms with growth suppressive functions as well as ?N dominant negative isoforms with oncogenic properties. Both isoforms undergo alternative splicing to generate multiple C-terminus variants, complicating molecular analysis of p63 function. Notably, ?-type C- terminus (C?) uniquely harbors a sterile ?-motif (SAM) protein-protein interaction domain and a transcription inhibitory (TI) domain. Their mutations and deletions in ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a unique form of p63-associated ectodermal dysplasia, highlight their functional importance. To further dissect the role of the C? domains, we have generated mice deficient in C? (p63C-/- mice). Notably, p63C-/- mice exhibit severe epidermal hypoplasia and fragility due to premature run down of the proliferative potential of EpiSCs. As we hypothesized that p63SAM interacting proteins were at least partially responsible for these effects, we screened for p63SAM interacting proteins and found that many of the identified proteins were involved in transcriptional and posttranscriptional activities. In Aim 1, we will further characterize p63SAM binding proteins by assessing their role in controlling the proliferative potential of EpiSCs and determine how deletion and AEC mutations in the individual C? domains impact their interactions. We will also interrogate the role of each C? domain in TA- and ?Np63? isoforms by reconstituting p63C-/- epidermal cells with wild type or C? domain mutant p63 isoforms. Finally, our preliminary data utilizing genome-wide array analysis coupled with clonogenic cultures suggest that the synergistic effects of Hgf and Igf2, regulated by the Wnt signaling mediator Dact1, are directly involved in EpiSC maintenance. In Aim 2, we will further assess the synergistic role of Hgf/Igf2 in regulating EpiSC self-renewal, proliferation and differentiation boh in vitro and in animal models and will determine if these growth factors regulate p63 levels to maintain the proliferative potential of EpiSCs. To further characterize the EpiSC niche, we have generated novel reporter mice to identify cells in which the Dact1 gene promoter is active, thereby allowing us to determine if they contribute to niche function in vivo. Together, these studies will provide unique insight into the intrinsic and extrinsic control of EpiSCs and will sere as a basis for development of novel strategies to improve the quality of EpiSCs for therapeutic transplantation and SC-directed gene therapy and for the treatment of wounds, burns and other degenerative epidermal disorders including cancer.